Time-Resolved Monitoring of Electrogenic Na+−Ca2+Exchange in the Isolated Cardiac Sarcolemma Vesicles by Using a Rapid-Response Fluorescent Probe

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Abstract
As a major Ca exit system in myocytes, the electrogenic Na+−Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation−contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+−Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 μM Cafree. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-state burst (210 s-1), followed by slow steady-state phase (≤5 s-1). Extravesicular (cytosolic) Ni inhibits both phases with an IC50 of 0.80 ± 0.24 mM. At an extravesicular pH of 6.0, the Nao-dependent Ca efflux is able to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+−Ca2+ exchange is not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry34, 10290−10297]. The M540 signal of Nao-dependent Ca efflux is lost when the extravesicular Cafree concentration drops to 0.2 μM. This effect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because the reaction of Nao-dependent Ca efflux utilizes intravesicular Ca as a substrate. These data suggest that in sarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of this putative regulatory site do not resemble the properties of the “slow” Ca regulatory mode, observed in electrophysiological studies. Under saturating ionic conditions, the Nao-dependent Ca efflux generates the initial rates of 21 mV/ms in the vesicles with a diameter of 3000−5000 Å. If a site density of 300−400 exchangers/μm2 and a vesicular surface of 0.5 μm2 are assumed, each vesicle may contain 150−200 exchanger molecules with a maximal turnover rate of 4000−5000 s-1. This upper limit for turnover (no matter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Ca entry in the cell under physiologically related conditions.